WO2021100875A1

WO2021100875A1 - Method and reagent for detecting bacterium involved with development of rheumatoid arthritis, and method and agent for determining presence or absence of predisposition to rheumatoid arthritis

Info

Publication number: WO2021100875A1
Application number: PCT/JP2020/043510
Authority: WO
Inventors: 潔竹田; 悠一前田; 小泉　聡司; 山下　誠; 史山▲崎▼; 林　幹朗
Original assignee: 国立大学法人大阪大学; 協和発酵バイオ株式会社
Priority date: 2019-11-20
Filing date: 2020-11-20
Publication date: 2021-05-27
Also published as: JPWO2021100875A1

Abstract

The objective of the present invention is to provide a means for identifying a bacterium associated with the development of rheumatoid arthritis and determining the possibility of contracting rheumatoid arthritis earlier and more easily using the bacterium. The present invention relates to a method and reagent for detecting a bacterium involved with the development of rheumatoid arthritis, and a method and agent for determining the presence or absence of a predisposition to rheumatoid arthritis.

Description

A method for detecting bacteria involved in the onset of rheumatoid arthritis, a reagent for detecting the bacteria, a method for determining the presence or absence of a predisposition to rheumatoid arthritis, and an agent for determining the presence or absence of the predisposition.

The present invention relates to a method for detecting a bacterium involved in the onset of rheumatoid arthritis, a reagent for detecting the bacterium, a method for determining the presence or absence of a predisposition to rheumatoid arthritis, and an agent for determining the presence or absence of the predisposition.

Rheumatoid arthritis (rheumatoid arthritis, RA) is a chronic systemic autoimmune disease that mainly affects joints and occurs in about 1% of the total population. Rheumatoid arthritis causes damage via cytokines, chemokines, and metalloproteases.

The onset of rheumatoid arthritis is usually insidious and often begins with systemic and joint symptoms. The pathology of rheumatoid arthritis is characterized by symmetrical inflammation of peripheral joints (eg, wrist and metacarpophalangeal joints), resulting in progressive destruction of joint structure, usually with systemic symptoms. .. Rheumatoid arthritis occurs 2-3 times more often in women than in men and can occur at any age, most often between the ages of 35 and 50, but can also occur in childhood or old age ( Non-Patent Document 1).

When the symptoms of rheumatoid arthritis worsen, the physical and mental distress to the patient increases and the cost of drugs used for treatment rises, so a mechanism for providing appropriate treatment at the appropriate time is required. Has been done.

Currently, the diagnosis of rheumatoid arthritis is comprehensively performed by scoring the number of swelling and painful joints and the presence or absence of rheumatoid factor and anti-CCP antibody in blood tests, but there is a possibility of getting rheumatoid arthritis earlier. There is a need for the development of biomarkers that can determine.

On the other hand, Non-Patent Document 2 discloses that the abundance ratio of Prevotella copper in the intestine increases in the early stage of rheumatoid arthritis.

Rheumatoid arthritis factors and anti-CCP antibodies that can be measured by the above blood tests, which are used for the diagnosis of rheumatoid arthritis, may be detected even in healthy subjects, but they are not detected in patients with rheumatoid arthritis. There is. In addition, Non-Patent Document 2 discloses that the abundance ratio of Prevotella copper in the intestine increases in the early stage of rheumatoid arthritis, but the relationship with the onset of rheumatoid arthritis is not clear.

An object of the present invention is to provide a means for identifying a bacterium associated with the onset of rheumatoid arthritis and using the bacterium to determine the possibility of contracting rheumatoid arthritis more early and easily.

The present inventors isolated Prevotella copper from stool samples of rheumatoid arthritis patients and healthy subjects, and Prevotella copper derived from rheumatoid arthritis patients (hereinafter abbreviated as RA-P.copri) is Prevotella copper derived from healthy subjects (hereinafter HC). It has been found that it has arthritis-inducing activity and causes aggravation of arthritis as compared with -P.copri). Furthermore, RA-P. copri and HC-P. As a result of analyzing the genome sequence of copri, RA-P. It was revealed that there is a region specific to the genome of copri. Based on the above findings, the present invention has been completed.

The present invention is as follows.
1. 1. A method for detecting a bacterium involved in the development of rheumatoid arthritis, which comprises a step of specifically detecting the nucleic acid sequence of any one of (1) to (3) below or a part of the sequence thereof.
(1) Nucleic acid sequence represented by SEQ ID NOs: 1 to 3 (2) Nucleic acid sequence having 95% or more homology with the nucleic acid sequence represented by SEQ ID NOs: 1 to 3 (3) The nucleic acid sequences represented by SEQ ID NOs: 1 to 3. 2. A nucleic acid sequence that hybridizes with a nucleic acid sequence or a nucleic acid sequence complementary to the nucleic acid sequence under stringent conditions. The detection method according to 1 above, wherein the bacterium involved in the development of rheumatoid arthritis belongs to the genus Prevotella.
3. 3. The detection method according to 2 above, wherein the bacterium belonging to the genus Prevotella is Prevotella copri.
4. The step of specifically detecting the nucleic acid sequence of any one of (1) to (3) or a part of the sequence thereof is carried out by a polymerase chain reaction using DNA derived from the feces of the subject as a template. The detection method according to any one of 3.
5. At least two types of primers used in the polymerase chain reaction contain a nucleic acid sequence consisting of at least 15 consecutive bases of the nucleic acid sequence shown in any one of SEQ ID NOs: 1 to 3 or a nucleic acid sequence complementary to the sequence. The detection method according to 4 above, which is an oligonucleotide to be used.
6. The detection method according to 5 above, wherein the primer is 1 oligonucleotide selected from the nucleic acid sequences represented by SEQ ID NOs: 4 to 19.
7. The detection according to any one of 1 to 3 above, wherein the step of specifically detecting the nucleic acid sequence or a part of the sequence is performed by metagenomic analysis using a next-generation sequencer targeting DNA derived from the feces of a subject. Method.
8. A method for determining the presence or absence of a predisposition to rheumatoid arthritis, which comprises a step of detecting bacteria involved in the onset of rheumatoid arthritis in the feces of a subject by the detection method according to any one of 1 to 7.
9. A reagent for detecting bacteria involved in the development of rheumatoid arthritis, which comprises a primer or probe for specifically detecting the nucleic acid sequence of any one of (1) to (3) below or a part of the sequence thereof.
(1) Nucleic acid sequence represented by SEQ ID NOs: 1 to 3 (2) Nucleic acid sequence having 95% or more homology with the nucleic acid sequence represented by SEQ ID NOs: 1 to 3 (3) The nucleic acid sequences represented by SEQ ID NOs: 1 to 3. 10. A nucleic acid sequence that hybridizes with a nucleic acid sequence or a nucleic acid sequence complementary to the nucleic acid sequence under stringent conditions. The primer or the probe is an oligonucleotide containing a nucleic acid sequence consisting of at least 15 consecutive bases of the nucleic acid sequence shown in any one of SEQ ID NOs: 1 to 3 or a nucleic acid sequence complementary to the sequence. 9. The detection reagent according to 9.
11. The detection reagent according to 10 above, wherein the primer or the probe is one oligonucleotide selected from the nucleic acid sequences represented by SEQ ID NOs: 4 to 19.
12. An agent for determining the presence or absence of a predisposition to rheumatoid arthritis, which comprises a primer or probe for specifically detecting the nucleic acid sequence of any one of (1) to (3) below or a part of the sequence thereof.
(1) Nucleic acid sequence represented by SEQ ID NOs: 1 to 3 (2) Nucleic acid sequence having 95% or more homology with the nucleic acid sequence represented by SEQ ID NOs: 1 to 3 (3) The nucleic acid sequences represented by SEQ ID NOs: 1 to 3. Nucleic acid sequence or a nucleic acid sequence that hybridizes with a nucleic acid sequence complementary to the nucleic acid sequence under stringent conditions 13. The primer or the probe is an oligonucleotide containing a nucleic acid sequence consisting of at least 15 consecutive bases of the nucleic acid sequence shown in any one of SEQ ID NOs: 1 to 3 or a nucleic acid sequence complementary to the sequence. The agent according to 12.
14. The agent according to 13 above, wherein the primer or probe is one oligonucleotide selected from the nucleic acid sequences represented by SEQ ID NOs: 4 to 19.

According to the method for detecting bacteria of the present invention and the reagent for detecting the bacteria, it is possible to specifically and easily detect the bacteria involved in the onset of rheumatoid arthritis even before the onset of rheumatoid arthritis. Further, according to the method for determining the presence or absence of a predisposition to rheumatoid arthritis and the agent for determining the presence or absence of the predisposition of the present invention, the possibility of suffering from rheumatoid arthritis is determined before the onset of rheumatoid arthritis, and it is appropriate at an appropriate time. It is possible to provide various treatments, which can be a new strategy from the viewpoint of preventive medicine.

FIG. 1 shows P.I. It is a figure which shows the result of having evaluated the arthritis-inducing activity by copy. FIG. 2 (A) shows the isolated RA-P. copri and HC-P. It is a figure which shows the result of having analyzed the genome structure of copy. FIG. 2 (B) shows RA-P. Of the primers used in the examples. It is a figure which shows the position in the genome of copri. FIG. 3 is a diagram showing the results of PCR performed using DNA extracted from feces of rheumatoid arthritis patients as a template. FIG. 4 is a diagram showing the results of PCR performed using DNA extracted from feces of a healthy subject as a template.

Hereinafter, the terms used in the present specification have meanings usually used in the art unless otherwise specified.

<1. Bacterial detection method>
The bacterium in the present invention is an intestinal bacterium having an activity of inducing arthritis. The activity that induces arthritis can be determined by increasing the arthritis score. The method for determining the increase in the arthritis score is not particularly limited, and specific examples thereof include the following methods.

In one embodiment, the bacterium in the present invention has the following nucleic acid sequence (1) or (2) or a partial sequence thereof.
(1) Nucleic acid sequence represented by SEQ ID NOs: 1 to 3 (2) Nucleic acid sequence having 95% or more homology with the nucleic acid sequence represented by SEQ ID NOs: 1 to 3.

Here, SEQ ID NOs: 1 to 3 have arthritis-inducing activity, RA-P. The nucleic acid sequence of the region specifically present in the genome of copri is shown. Homology between nucleic acid sequences is calculated using the homology search program BLAST. When the bacterium has a nucleic acid sequence having 95% or more homology with the nucleic acid sequence represented by SEQ ID NOs: 1 to 3, RA-P. Similar to copri, it is highly likely that it has the activity of inducing arthritis.

As a method for determining the activity of inducing arthritis by increasing the arthritis score, the method described in Takeichi Kakimoto et al., New Chemistry Experiment Course 12, Molecular Immunology II, pp. 360-372, Tokyo Kagaku Dojin (1989) can be mentioned. Be done. Specifically, the following method can be mentioned. First, an arthritis model mouse (eg, DBA / 1j mouse) is administered with an antibiotic (eg, ampicillin, neomycin, metronidazole and vancomycin) for several days to kill the intestinal bacteria. Later, the test bacteria are divided into a group to be administered or a healthy subject-derived bacterium to be administered (control group), and each bacterium is orally administered for several consecutive days (for example, 5 days). Feces are collected 3 days after the final administration to confirm that each bacterium has settled in the mice, and an emulsion containing a mixture of type II collagen and an adjuvant is intradermally administered to each group of mice. After 3 weeks, as a booster, an emulsion of a mixture of type II collagen and an adjuvant is re-administered and the arthritis score is evaluated over time. If the arthritis score is statistically significantly higher than that of the control group 5 to 8 weeks after the administration of the second emulsion, it can be determined that the test bacterium has the activity of inducing rheumatoid arthritis.

The bacterium in the present invention is Prevotella copri, which is a gram-negative anaerobic bacterium. Whether or not the bacterium isolated from the isolation source is Prevotella copper can be determined, for example, by comparing the nucleotide sequence data of the 16S ribosomal RNA gene with the sequence data of a known species and performing phylogenetic analysis.

In the present specification, "detection of bacteria" includes not only determining the presence or absence of bacterial cells, but also quantifying the abundance thereof.

When detecting bacteria, collect all DNA from the sample. Examples of the type of the sample include feces or intestinal contents of a subject (healthy subject, rheumatoid arthritis patient or individual suspected of having rheumatoid arthritis), isolated / cultured bacteria and the like. It is not limited to these. A method for isolating / purifying DNA from a sample is known in the art, and can be carried out by, for example, extraction with phenol / chloroform, extraction with a commercially available DNA extraction reagent, purification with a commercially available column kit, or the like.

The DNA recovered from the sample is dissolved in an appropriate buffer solution such as TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and used for the detection method of the present invention.

The method for detecting a bacterium of the present invention is characterized by comprising a step of specifically detecting the nucleic acid sequence of any one of (1) to (3) below or a part of the sequence thereof.
(1) Nucleic acid sequence represented by SEQ ID NOs: 1 to 3 (2) Nucleic acid sequence having 95% or more homology with the nucleic acid sequence represented by SEQ ID NOs: 1 to 3 (3) The nucleic acid sequences represented by SEQ ID NOs: 1 to 3. Nucleic acid sequence or a nucleic acid sequence that hybridizes with a nucleic acid sequence complementary to the nucleic acid sequence under stringent conditions

Examples of the nucleic acid sequence that hybridizes under stringent conditions include a nucleic acid sequence having a certain degree of sequence identity with the nucleic acid sequence used as a probe. For example, a nucleic acid sequence having at least 60% or more homology with the target nucleic acid sequence, preferably a nucleic acid sequence having preferably 80% or more homology, still more preferably a nucleic acid sequence having 90% or more homology, most preferably. Examples include genes having 95% or more homology. Further, for example, assuming that the number of nucleic acids in the nucleic acid sequence is 100 as one unit, in the nucleic acid sequence of the target gene, 1 to several, preferably 1 to 40, preferably 1 to 35 per unit. , Preferably 1 to 30, preferably 1 to 25, preferably 1 to 20, more preferably 1 to 15, still more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 Or 10 nucleic acid sequences, more preferably 1, 2, 3, 4 or 5 nucleic acid deletions, substitutions, additions and the like.

"Nucleic acid deletion" means that the nucleic acid in the sequence is missing or missing, "nucleic acid substitution" means that the nucleic acid in the sequence has been replaced by another nucleic acid, and "nucleic acid substitution". "Addition" means that new nucleic acid has been added for insertion.

In one aspect, the detection method of the present invention preferably uses a primer or probe capable of specifically detecting the nucleic acid sequence of any one of (1) to (3) above or a part of the sequence thereof.

The primer may be any as long as it is designed so that a part of the region of the nucleic acid sequence can be specifically PCR-amplified. The nucleic acid sequence of any one of (1) to (3) above or a part of the sequence thereof has arthritis-inducing activity in the present invention, RA-P. A nucleic acid sequence present in the genome of copri.

Here, "specifically" means that the primer PCR-amplifies a part of the region of the nucleic acid sequence of any one of the above (1) to (3), but any of the above (1) to (3). This means that a nucleic acid sequence that does not contain one nucleic acid sequence or a part of the sequence thereof is not PCR-amplified.

As the primer, for example, a continuous, preferably 15 to 50 bases, more preferably 18 to 30 bases selected from the nucleic acid sequence represented by any one of SEQ ID NOs: 1 to 3 or the nucleic acid sequence complementary to the sequence. Examples thereof include a pair of oligonucleotides which are a combination of oligonucleotides containing a nucleic acid sequence of bases, and the fragment length of the nucleic acid amplified by them is preferably 50 to 1,000 bases, more preferably 100 to 500 bases.

Specific examples of preferred primers include combinations of oligonucleotides selected from the nucleic acid sequences represented by SEQ ID NOs: 4 to 19.

The temperature setting, reaction time and number of cycles in PCR can be appropriately set according to the amount of template DNA to be used, the type of primer, and the like. The annealing temperature in PCR can be appropriately set based on the GC content of the primer. For example, using the genomic DNA of a bacterium as a template and an oligonucleotide consisting of the nucleic acid sequence represented by SEQ ID NO: 4 and an oligonucleotide consisting of the nucleic acid sequence represented by SEQ ID NO: 5 as primers, 95 ° C. for 30 seconds, 55. The reaction can be carried out under the condition that the temperature is 30 seconds and the temperature is 72 ° C. for 45 seconds for 30 cycles.

The probe is contained in the nucleic acid sequence represented by SEQ ID NO: 1 to 3 or a nucleic acid sequence complementary to the nucleic acid sequence, preferably 15 bases or more, more preferably 18 to 500 bases, still more preferably 18 to 200 bases. , Particularly preferably, an oligonucleotide that hybridizes to a contiguous nucleic acid sequence of 18 to 50 bases under stringent conditions.

Hybridization should be performed according to a method known per se or a method similar thereto, for example, the method described in Molecular Cloning 2nd Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Can be done. As stringent conditions, for example, after a hybridization reaction at 45 ° C. in 6 × SSC (sodium chloride / sodium citrate), one or more times at 65 ° C. in 0.2 × SSC / 0.1% SDS. Cleaning etc. can be mentioned. By appropriately changing the salt concentration of the hybridization solution, the temperature of the hybridization reaction, the probe concentration, the length of the probe, the number of mismatches, the time of the hybridization reaction, the salt concentration of the washing solution, the washing temperature, etc. , Can be easily adjusted to the desired stringency.

The length of the probe is preferably 15 bases or more, more preferably 18 to 500 bases, still more preferably 18 to 200 bases, and particularly preferably 18 to 50 bases.

From the viewpoint of specificity, the probe preferably includes an oligonucleotide that hybridizes to the nucleic acid sequence represented by any one of SEQ ID NOs: 4 to 19 or its complementary sequence. More preferably, an oligonucleotide containing a continuous subsequence of 15 to 50 bases of the nucleic acid sequence represented by any one of SEQ ID NOs: 4 to 19 or a complementary sequence thereof can be mentioned.

Specific examples of preferable probes include oligonucleotides selected from the nucleic acid sequences represented by SEQ ID NOs: 4 to 19.

The primer or probe may contain an additional sequence (nucleic acid sequence that is not complementary to the polynucleotide to be detected) as long as it does not interfere with specific detection.

The oligonucleotide used as the primer or probe may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In the case of ribonucleic acid, the thymidine residue (T) in the nucleotide sequence is appropriately read as the uridine residue (U). Further, it may be a DNA containing a uridine residue synthesized by changing T at an arbitrary position to U. Similarly, it may be RNA containing a thymidine residue synthesized by changing U at an arbitrary position to T. In addition, point mutations such as deletions, insertions or substitutions, and modified nucleotides may be present in the oligonucleotide as long as the specificity of hybridization is not reduced.

Primers or probes can also be suitable labeling agents such as radioisotopes (eg ¹²⁵ I, ¹³¹ I, ³ H, ¹⁴ C, ³² P, ³³ P, ³⁵ S, etc.), enzymes (eg β-galactosidase, etc.). , Β-Glucosidase, alkaline phosphatase, peroxidase, malic acid dehydrogenase, etc.), fluorescent substances (eg, fluorescamine, fluoressen isothiocyanate, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.) , Biotin or the like.

The oligonucleotide used as the primer or probe can be chemically synthesized, for example, using a general-purpose DNA synthesizer. Oligonucleotides may be synthesized using any of the other methods well known in the art.

Includes PCR using the DNA recovered from the sample as a template using the primers contained in the detection reagent of the present invention. The obtained PCR product is separated by electrophoresis (for example, agarose gel electrophoresis, polyacrylamide gel electrophoresis, etc.). After electrophoresis, the gel is stained with a stain solution known per se, such as an ethidium bromide solution, and the PCR product is detected using a transilluminator or the like. Then, the presence / absence and abundance of the bacterium of the present invention in the sample are determined using the presence / absence and amount of the specific PCR product as an index.

The PCR used in the detection method of the present invention may be quantitative PCR. Quantitative PCR can be performed by known methods, but two analytical methods are known. The first is to analyze the reaction product amount during the exponential growth period by utilizing the feature that the reaction product increases exponentially up to a certain amount in the PCR reaction and then reaches the plateau, and the initial template amount. Is a method of calculating. The second method is to determine the number of PCR cycles (Ct) in which the amount of reaction product exceeds a certain value (threshold) by monitoring the reaction product in real time. In each analysis method, it is necessary to perform PCR by changing the amount of DNA at a known concentration, analyze the reaction product at each cycle number, and determine a quantitative PCR cycle number range from the kinetics. Based on the results, the abundance of the target gene in an unknown sample is estimated. Thereby, the abundance of the bacterium of the present invention in the sample can be quantified, and when it is estimated that even one copy of the target gene is contained in the test sample, it can be determined that the bacterium of the present invention is present.

In one aspect, the detection method of the present invention includes a step of bringing the probe contained in the detection reagent of the present invention into contact with all the DNA in the sample. The contact condition is that the probe is RA-P. It is appropriately set to hybridize with a nucleic acid sequence specifically present in the genome of copri to form a nucleic acid complex. The complex is then detected as an indicator of the presence of the bacterium of the present invention.

When a probe is used, it can be carried out by various known hybridization techniques [for example, fluorescence in situ hybridization method [Fluorescence In situ hybridization (hereinafter abbreviated as FISH)]]. In the FISH method, the probe invades the bacterial cytoplasm and is present in the RA-P. It hybridizes to the nucleic acid sequence present in the genome of copri under appropriate hybridization conditions. At this time, by labeling the probe with a radioactive isotope, a fluorescent substance [for example, fluorescein isothiocyanate (FITC), TAMRA, Cy3, Cy5, etc.], a chemical luminescent substance, etc., a specific hybridization phenomenon can be appropriately observed. It can be monitored by techniques (eg, autoradiography, fluorescence microscopy, flow cytometry, etc.). For example, if the probe is labeled with a radioisotope, the assay is performed by a method such as autoradiography, and if the probe is labeled with a fluorescent substance, the assay is performed with a fluorescence microscope or the like, and the chemiluminescent substance is used. If labeled, analysis using a photosensitive film or digital analysis using a CCD camera can be performed. Thereby, bacteria in the sample can be detected.

In one aspect, the detection method of the present invention includes metagenomic analysis by a next-generation sequencer. The next-generation sequencer is a base sequence analysis (decoding) device that dramatically improves the analysis speed by parallel processing, and is called a fluorescent capillary sequencer (called "first-generation sequencer") that uses the Sanger sequencing method. Make a contrast. By using metagenomic analysis using a next-generation sequencer, it is possible to analyze all bacterial species that make up the intestinal flora and their abundance ratios. The method is not particularly limited as long as it is a method for clarifying the diversity of the intestinal flora and the abundance ratio of specific bacteria constituting the intestinal flora.

As commercially available next-generation sequencers, for example, Illumina (for example, HiSeq 2500, HiSeq X Ten, NextSeq 500), Roche (454) (for example, GS FLX + system), Life Technologies (for example, 5500xlSOLID), For example, various next-generation sequencers sold by manufacturers such as Proton Sequencer) can be mentioned. Not limited to the existing massively parallel next-generation sequencer, the next-generation sequencer that will be developed and released in the future may be used.

<2. Bacterial detection reagent>
The detection reagent of the present invention is the RA-P. By detecting the nucleic acid sequence present in the genome of copri, the presence of the bacterium of the present invention in the sample is detected. The detection reagent of the present invention can specifically detect the nucleic acid sequence of any one of the following (1) to (3), <1. Bacterial detection method> includes the above-mentioned primers or probes.
(1) Nucleic acid sequence represented by SEQ ID NOs: 1 to 3 (2) Nucleic acid sequence having 95% or more homology with the nucleic acid sequence represented by SEQ ID NOs: 1 to 3 (3) The nucleic acid sequences represented by SEQ ID NOs: 1 to 3. Nucleic acid sequence or a nucleic acid sequence that hybridizes with a nucleic acid sequence complementary to the nucleic acid sequence under stringent conditions

The detection reagent of the present invention further contains nucleic acid synthase (for example, DNA polymerase, RNA polymerase, reverse transcriptase, etc.), other enzymes, substrates corresponding to the enzymes (for example, dNTP, rNTP, etc.) as other components. It may be included. It may also contain a label detection substance, a buffer solution, or the like.

If the detection reagent of the present invention is used, the presence or absence of bacteria that induce rheumatoid arthritis in the sample can be easily determined in a short time, which is useful for diagnosing the predisposition to rheumatoid arthritis.

<3. How to determine the presence or absence of a predisposition to rheumatoid arthritis>
As shown in Examples described later, the bacterium of the present invention having the nucleic acid sequences represented by SEQ ID NOs: 1 to 3 was detected in the feces of a person suffering from rheumatoid arthritis. Considering that the bacterium in the present invention has an arthritis-inducing activity, the presence of the bacterium in the present invention in the intestine of a subject is determined regardless of whether or not the subject has already developed rheumatoid arthritis. , Shows that it has a predisposition to develop rheumatoid arthritis (predisposition to rheumatoid arthritis).

Therefore, <1. Bacterial detection method> By detecting the bacteria involved in the onset of rheumatoid arthritis in the feces of the subject, the presence or absence of a predisposition to rheumatoid arthritis can be determined. That is, the present invention provides a method for determining the presence or absence of a predisposition to rheumatoid arthritis, which comprises detecting the bacteria of the present invention in the feces of a subject.

The bacterium in the present invention uses the feces of the subject as a sample, and <1. Bacterial detection method> can be detected by the detection method described. When the bacterium of the present invention is detected in the feces of a subject, it can be determined that the subject has a predisposition to rheumatoid arthritis and the risk of developing rheumatoid arthritis is relatively high. On the contrary, when the bacterium in the present invention is not detected, it can be determined that the subject has a relatively low risk of developing rheumatoid arthritis.

<4. Agents for determining the predisposition to rheumatoid arthritis>
The present invention provides an agent for determining the presence or absence of a predisposition to rheumatoid arthritis. The agent of the present invention determines the presence or absence of a predisposition to rheumatoid arthritis by detecting the presence of the bacterium in the present invention in the feces of a subject. Therefore, the agent of the present invention is preferably <2. Bacterial Detecting Reagents> contains the detection reagents set forth, and more preferably contains a primer set of oligonucleotides selected from the nucleic acid sequences represented by SEQ ID NOs: 4-19. By using the agent of the present invention, the presence or absence of a predisposition to rheumatoid arthritis can be easily determined by the above-mentioned method.

Examples are shown below, but the present invention is not limited to the following examples.

1. 1. Isolation of Prevotella copri Prevotella copri (hereinafter abbreviated as P. copli) was isolated from the feces of rheumatoid arthritis patients and healthy subjects by the following method. _{10 mL of PBS (1.37 mM NaCl, 10 mM Na 2} HPO ₄ , 2.7 mM KCl, 1.76 mM KH ₂ PO ₄ , pH 7) in which 1 g of feces stored at -80 ° C was thawed and anaerobically replaced in an anaerobic chamber. Suspended in 4). The suspension was prepared with 10-fold serial dilutions in PBS ⁽¹⁰ 4 ~ 10 ⁸ dilution). 50 μL of each diluted solution was applied to a Colombian 5% sheep blood agar medium (manufactured by Becton Dickinson, Japan) and cultured anaerobically at 37 ° C. for 2 days. The appearing colonies were pecked with a toothpick, and PCR was performed using Prevotella primers (g-Prevo-F; SEQ ID NO: 20 and g-Prevo-R; SEQ ID NO: 21) using the cells of each colony as a template.

PCR was performed with the following reaction composition. The "one pinch of toothpick" in the following reaction composition means the amount of bacterial cells attached to the toothpick by poking the colony once with the toothpick.
Colony cells 1 pinch 10xPCR buffer 5.0 μL
2 mM dNTP 5.0 μL
rTaq (manufactured by Toyobo Life Science Co., Ltd.) 0.2 μL
Primer g-Prevo-F (100 pM) 0.2 μL
Primer g-Prevo-R (100 pM) 0.2 μL
Deionized water 39.4 μL

The PCR reaction was carried out under the conditions of 35 cycles of 95 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 90 seconds. The reaction solution was subjected to electrophoresis using a 2% agarose gel, and amplification of the fragments was confirmed.
The colonies from which the amplification products were obtained were again applied to Colombian 5% sheep blood agar and cultured anaerobically at 37 ° C. for 2 days. From the appearing colonies, PCR was performed using the universal primers (8F; SEQ ID NOs: 22 and 15R; SEQ ID NO: 23) of the 16S rRNA gene in the same manner as above. It was confirmed by electrophoresis that the PCR product was amplified. The PCR product was purified by a conventional method, and PCR using the purified DNA as a template and sequencing primers (SEQ ID NOs: 24-28) was performed with the following reaction composition.

Purified DNA 4.5 μL
5x sequence buffer 2.0 μL
BigDye v3.1
(Manufactured by Thermo Fisher) 1.0 μL
Sequencer primer (10 μM) 1.0 μL
Deionized water 1.5 μL

The PCR reaction was carried out under the conditions of performing 25 cycles of 96 ° C. for 10 seconds, 50 ° C. for 5 seconds, and 60 ° C. for 4 minutes. After the reaction solution was precipitated with ethanol, the base sequence was determined by a sequencer. The obtained 16S rRNA sequence was analyzed by BLAST search, and a bacterium that matched the sequence of Prevotella copper was identified as Prevotella copper.

The strain identified as Prevotella copper was anaerobically cultured in GAM liquid medium (manufactured by Nissui Pharmaceutical Co., Ltd.) at 37 ° C. for 1 to 2 days. The culture was mixed with an equal volume of sterilized 80% glycerol solution and stored at -80 ° C.

2. P. Evaluation of arthritis-inducing activity by copy 1. P. et al. From an arthritis patient isolated in Copri (hereinafter abbreviated as RA-P.copri) and P. An experiment was conducted in which copri (hereinafter abbreviated as HC-P. copli) was anaerobically cultured and administered to arthritis model mice to evaluate susceptibility.

6 to 7 week old DBA / 1j male mice (Charles River Japan) were administered with 4 types of antibiotics (ampicillin 500 mg / L, neomycin 500 mg / L, metronidazole 500 mg / L, vancomycin 250 mg / L) for 5 days. Killed intestinal bacteria. RA-P. Cultured from 2 days after the end of antibiotic administration. copri and HC-P. copri was orally administered for 5 consecutive days. The administered cells were cultured in a GAM liquid medium for 24 hours in an anaerobic chamber, and then suspended in PBS so that the OD600 value became 1.5.

Three days after the last administration day, feces were collected and DNA was extracted, and P.I. P.I., which is contained in feces by the qPCR method using a copper-specific primer. The amount of bacteria in copri was quantified, and each P. It was confirmed that copri was established in the mouse. Two days after the completion of bacterial cell administration, an emulsion (final) in which bovine type II collagen (collagen technology workshop K41 TypeII) and adjuvant [Friend's complete adjuvant (BD; DF0638-60-7)] were mixed 1: 1. A collagen concentration of 1 mg / mL) was prepared, and 100 μL of each was intradermally administered to DBA / 1j mice. After 3 weeks, as a booster, 100 μL of the emulsion prepared in the same manner was re-administered.

After that, the arthritis score of the mice was evaluated over time every other week. The evaluation method for arthritis in mice is 0 point; normal, 1 point; swelling or redness in 1 joint, 2 points; swelling or redness in 2 or more joints, 3 points; all joints. There was swelling or redness, and the score was 4 points; deformed / ankylosis, with a maximum of 16 points. The results are shown in FIG.

As shown in FIG. 1, 5-8 weeks after the administration of the second emulsion, RA-P. Mice treated with copri were HC-P. Severe arthritis was observed as compared with the mice treated with copri. From this result, RA-P. It has been shown that copri may act as an exacerbating factor for rheumatoid arthritis.

3. 3. Genome analysis of isolated strains 1. RAP isolated in. copri and HC-P. For copy, DNA was extracted using DNeasy PowerSoil Kit (manufactured by QIAGEN). The whole genome sequence of each strain was a long read sequence by MinION (manufactured by Oxford Nanopore) and a short read sequence by MiSeq (manufactured by Illumina).

Using the obtained long read and short read, a hybrid assembly method using a Unicycler was carried out to construct a full-length genome. By performing a homology search on the genome sequences of each of the constructed strains using blastn with reference to each other's sequences, regions in which there is duplication between the genomes were obtained. By visualizing the region obtained by the blastn search using Circos, a region in which a defect was found only in the strain was found.

In addition, the phylogenetic relationship was estimated based on the genetic information by creating the core genome. First, after performing gene annotation using RAST from the genome sequence, the same gene was searched for by performing a homology search using blastp on the amino acid sequence of each coding region. At that time, genes commonly found among the strains were extracted after limiting the amino acid homology to 95% or more and the length ratio (shorter / longer) of 0.8 or more. The common genes were aligned by maft for each gene, and then the amino acid sequences were bound for each strain in the same order to prepare a core genome sequence. The distance between the core genomes was calculated by Fast Tree and used to estimate the phylogenetic relationship.

As a result, RA-P. The existence of a region specific to the genome of copri was revealed. The results are shown in FIG. 2 (A). RA-P. The sequences of regions 1 to 3 specific to the genome of copri are shown in SEQ ID NOs: 1 to 3, respectively.

4. Detection by PCR After well suspending 1 g of feces of healthy subjects and patients with rheumatoid arthritis in 10 mL of PBS, DNA was extracted from 200 μL of the suspension by the method described in Maeda et al. (Arthritis Rheumatol, 2016; 68: 2646-2661). Extracted.

PCR was performed with the following reaction composition.
Fecal extract DNA (50 ng / μL) 1.0 μL
2xHSGoTaq (manufactured by Promega) 7.5 μL
Primer Fw (10 μM) 1.0 μL
Primer Rv (10 μM) 1.0 μL
Nuclease-Free Water 4.5 μL

The PCR reaction was carried out under the conditions of performing 30 cycles of 95 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 45 seconds. The combination of A to H primers used in each PCR reaction, amplification length and RA-P. The position of copri in the genome is shown in Table 1. FIG. 2 (B) shows RA-P. Of the primers used in the examples. It is a figure which shows the position in the genome of copri. After the reaction, each reaction solution was subjected to electrophoresis using a 2% agarose gel, and amplification of the fragments was confirmed.

As shown in Table 1, amplification of a DNA fragment of the desired size was confirmed only when DNA extracted from feces derived from patients with rheumatoid arthritis was used. From the above, by PCR, RA-P. It was found that the region specific to the genome of copri was amplified.

Although the present invention has been described in detail using specific embodiments, it will be apparent to those skilled in the art that various modifications and modifications can be made without departing from the intent and scope of the invention. This application is based on a Japanese patent application (Japanese Patent Application No. 2019-209926) filed on November 20, 2019, and the entire application is incorporated by reference.

Claims

A method for detecting a bacterium involved in the development of rheumatoid arthritis, which comprises a step of specifically detecting a nucleic acid sequence of any one of (1) to (3) below or a part of the sequence thereof.
(1) Nucleic acid sequence represented by SEQ ID NOs: 1 to 3 (2) Nucleic acid sequence having 95% or more homology with the nucleic acid sequence represented by SEQ ID NOs: 1 to 3 (3) The nucleic acid sequences represented by SEQ ID NOs: 1 to 3. Nucleic acid sequence or a nucleic acid sequence that hybridizes with a nucleic acid sequence complementary to the nucleic acid sequence under stringent conditions
The detection method according to claim 1, wherein the bacterium involved in the onset of rheumatoid arthritis belongs to the genus Prevotella.
The detection method according to claim 2, wherein the bacterium belonging to the genus Prevotella is Prevotella copper.
1. The step of specifically detecting the nucleic acid sequence of any one of (1) to (3) or a part of the sequence thereof is carried out by a polymerase chain reaction using DNA derived from the feces of a subject as a template. The detection method according to any one of 3 to 3.
At least two types of primers used in the polymerase chain reaction contain a nucleic acid sequence consisting of at least 15 consecutive bases of the nucleic acid sequence shown in any one of SEQ ID NOs: 1 to 3 or a nucleic acid sequence complementary to the sequence. The detection method according to claim 4, wherein the oligonucleotide is an oligonucleotide.
The detection method according to claim 5, wherein the primer is one oligonucleotide selected from the nucleic acid sequences represented by SEQ ID NOs: 4 to 19.
The step according to any one of claims 1 to 3, wherein the step of specifically detecting the nucleic acid sequence or a part of the sequence thereof is performed by metagenomic analysis by a next-generation sequencer targeting DNA derived from the feces of a subject. Detection method.
A method for determining the presence or absence of a predisposition to rheumatoid arthritis, which comprises a step of detecting bacteria involved in the onset of rheumatoid arthritis in the feces of a subject by the detection method according to any one of claims 1 to 7.
A reagent for detecting bacteria involved in the development of rheumatoid arthritis, which comprises a primer or probe for specifically detecting the nucleic acid sequence of any one of (1) to (3) below or a part of the sequence thereof.
(1) Nucleic acid sequence represented by SEQ ID NOs: 1 to 3 (2) Nucleic acid sequence having 95% or more homology with the nucleic acid sequence represented by SEQ ID NOs: 1 to 3 (3) The nucleic acid sequences represented by SEQ ID NOs: 1 to 3. Nucleic acid sequence or a nucleic acid sequence that hybridizes with a nucleic acid sequence complementary to the nucleic acid sequence under stringent conditions
The primer or the probe is an oligonucleotide containing a nucleic acid sequence consisting of at least 15 consecutive bases of the nucleic acid sequence shown in any one of SEQ ID NOs: 1 to 3 or a nucleic acid sequence complementary to the sequence. The detection reagent according to claim 9.
The detection reagent according to claim 10, wherein the primer or the probe is one oligonucleotide selected from the nucleic acid sequences represented by SEQ ID NOs: 4 to 19.
An agent for determining the presence or absence of a predisposition to rheumatoid arthritis, which comprises a primer or probe for specifically detecting the nucleic acid sequence of any one of (1) to (3) below or a part of the sequence thereof.
(1) Nucleic acid sequence represented by SEQ ID NOs: 1 to 3 (2) Nucleic acid sequence having 95% or more homology with the nucleic acid sequence represented by SEQ ID NOs: 1 to 3 (3) The nucleic acid sequences represented by SEQ ID NOs: 1 to 3. Nucleic acid sequence or a nucleic acid sequence that hybridizes with a nucleic acid sequence complementary to the nucleic acid sequence under stringent conditions
The primer or the probe is an oligonucleotide containing a nucleic acid sequence consisting of at least 15 consecutive bases of the nucleic acid sequence shown in any one of SEQ ID NOs: 1 to 3 or a nucleic acid sequence complementary to the sequence. The agent according to claim 12.
The agent according to claim 13, wherein the primer or the probe is one oligonucleotide selected from the nucleic acid sequences represented by SEQ ID NOs: 4 to 19.